Liquid phase peptide synthesis of KL-4 pulmonary surfactant

ABSTRACT

The invention realtes to improved liquid phase processes for the preparation of the 21 residue protein component, (Lys-Leu 4 ) 4 -Lys, of the pulmonary surfactant KL-4. These process are amenable to large scale synthesis and one process employs a method of saponifying an ester which reduces the inherent racemization of the α-carbon.

FIELD OF THE INVENTION

[0001] The present invention relates to the field of peptide synthesesand intermediates useful therein. More particularly, the inventionrelates to the synthesis of the polypeptide component of KL-4, asynthetic pulmonary surfactant.

BACKGROUND

[0002] The epithelium of mammalian lungs are lined with an endogenouspulmonary surfactant (PS) which facilitates breathing by aiding thetransport of oxygen across the lung air-liquid interface. A deficiencyin this surfactant is the primary cause of neonatal respiratory distresssyndrome (RDS) and is linked to RDS in adults as well. Native PS is amixture of lipids and proteins, and although its exact composition isunknown, researchers have prepared a number of exogenous surfactantswhich are useful in the treatment of RDS in pre-term infants. KL-4 is anexample of an exogenous surfactant which is useful in the treatment ofRDS as disclosed in U.S. Pat. Nos. 5,164,369, 5,260,273 and 5,407,914,hereby incorporated by reference.

[0003] KL-4 is a mixture of a pharmaceutically acceptable phospholipidand a 21 residue polypeptide, (L-lysine-(L-leucine)₄)₄L-lysine. (SEQ. IDNo. 1) As disclosed in the aforementioned patents, this peptide wasprepared by solid phase synthesis and recombinant DNA techniques. Thesolid phase synthesis comprises sequential addition of one or more aminoacid residues coupled with suitable protection of amino or carboxylgroups. Although this process is effective, it is not amenable to thelarge scale synthesis necessary to manufacture a drug substance.

[0004] The object of the present invention is the production of thepeptide component of KL₄, namely (Lys-Leu₄)₄Lys (SEQ. ID No. 1) by aliquid phase peptide synthesis (“LPPS”). Unlike the methods disclosed inthe aforementioned patents, this process is amenable to large scalesynthesis.

[0005] An additional embodiment of the invention concerns thedeprotection of the carboxy terminus of a peptide which was protected asan ester. Most peptide syntheses require manipulation of carboxyl andamino protecting groups. Typically, terminal carboxyl groups areprotected as their ester derivative. However deprotection methods may beaccompanied by racemization of the a-carbon; a problem that iscompounded as the length of the peptide increases. In several steps ofthe synthesis of (Lys-Leu₄)₄Lys, an ester protected carboxl group isdeprotected. As with most biomimetic products, the configuration of thepeptide is crucial and the active configuration of the residues of(Lys-Leu₄)₄Lys is “L”. This invention discloses a method of deprotectinga peptide's ester protected carboxyl group which reduces the amount ofracemized product. Although this method is applied in the synthesis of(Lys-Leu₄)₄Lys, it may be used in the synthesis of other peptides asillustrated hereinafter.

SUMMARY OF THE INVENTION

[0006] The invention relates to improved LPPS processes for thepreparation of the 21 residue protein component of the pulmonarysurfactant, KL-4, which is amenable to large scale synthesis. The firstmethod uses three peptide fragments:

[0007] The process starts off with a 3-residue right hand fragment whichis successively reacted with the 5-residue body fragment to build an18-amino acid fragment of the formulaH-Leu-Leu-(Lys(Z)-Leu₄)₃-Lys(Z)-OBzl (SEQ. ID No. 3). This 18-amino acidfragment is then reacted with the 3-residue right hand fragment to formthe final 21 residue protein of the present invention.

[0008] In the second method, a convergent synthesis is employed in whichan 8-residue protected polypeptide of the formula:Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 4) is preparedand saponified with tetraalkylammonium hydroxide. The saponified peptideis then reacted with a 13-residue peptide of the formulaH-Leu-Leu-(Lys(Z)-Leu₄)₂-Lys-OBzl (SEQ. ID No. 5) to yield the protected21-amino acid peptide. Removal of the protecting group by reaction witha suitable acid yields the final KL-4 polypeptide. This convergentmethod exhibits certain advantages in solubility and control overunwanted by-products, which makes the method particularly suitable tolarge scale synthesis.

[0009] In another aspect of the present invention is a novel method forthe deprotection of the carboxy terminus of a peptide protected as anester. This method comprises saponifying the protected peptide withtetraalkylammonium hydroxide reagent in a suitable solvent. This processprovides a method of deprotecting the peptide which reduces the amountof racemization at the α-carbon.

DETAILED DESCRIPTION OF THE INVENTION

[0010] Herein, the amino acid nomenclature corresponds to standardconventions where: L-leucine is “L” or “Leu”, L-lysine is “K” or “Lys”,L-alanine is “A” or “Ala”, benzyloxycarbonyl is “Z” or CBZ,D-(1-naphthyl)alanine is “D-Nal”, 4-chlorophenylalanine is “D-Cal”,L-serine is “L-Ser” and D-3-pyridylalanine is “D-Pal”. In addition allamino acid residue sequences are represented by formulae whose left toright orientation is in the conventional direction of amino-terminus tocarboxy-terminus. A dash at the beginning or end of the sequenceindicates a bond to a radical such as H, OH or OBzl; and a dash in themiddle of the sequence indicates a convential amide bond. Otherabbreviations and symbols are as follows: DMF is N,N-dimethylformamide,MeOH is methanol, HOBT is 1-hydroxybenzotriazole, THF istetrahydrofuran, DCC is 1,3 dicyclohexylcarbodiimide, EtOH is ethanol,iPrOH is isopropanol, HBTU is N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium-hexafluororphosphate), DIPEA isN,N-diisopropylethylamine, NMP is 1-methyl-2-pyrrolidone, HOOBT is3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, WSCDI (water solublecarbodiimide) is 1-(dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride and DIC is 1,3-diisopropylcarbodiimide. The term “alkyl”includes straight and branched groups; the term “hydroxide” includesGroup I metal hydroxides (NaOH, LiOH and the like) as well astetraalkylammonium hydroxides; the term “salts” includestetraalkylammonium halides and tetrahalo borates and “Ac” is acetyl.

[0011] The synthesis of (Lys-Leu₄)₄Lys (SEQ. ID No. 1) in accordancewith the present invention, follows two pathways. Procedure A uses threepeptides fragments: a 3-residue left-hand (amino-end), a three residueright hand (carboxy terminus) and a five residue body fragment toconstruct the molecule.

[0012] As illustrated in Scheme A, H-Leu-Leu-Lys(Z)-OBzl andBoc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) are reacted together inthe presence of a peptide coupling agent and an inert solvent at about0° C. to room temperature to giveBoc-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl. (SEQ. ID No. 6) Examplesof suitable peptide coupling agents include: DCC, DIC, HBTU, WSCDI,HOBT, HOOBt, where the preferred agents are HOOBT and HTBU. Solvents arechosen for their compatibility with the chosen coupling agent. Suitablesolvents include DMF, THF, NMP and acetonitrile, where a mixture of DMFand acetonitrile is preferred. If the acid salt of H-Leu-Leu-Lys(Z)-OBzlis used, this peptide is neutralized with an organic base. The Bocprotecting group is cleaved by treatingBoc-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6) with anacid at about −20 to 0° C. under an inert atmosphere. Although a solventmay be used with a gaseous acid, such as HCl, the preferred method usesneat trifluoroacetic acid at about 0° C.

[0013] H-Leu-Leu-Lys(Z)-Leu₄-Lys(Z)-OBzl (SEQ. ID No. 6) is treated witha peptide coupling agent, an organic base andBoc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) in an inert solvent atabout −4 to 10° C. to give Boc-Leu-Leu-(Lys(Z)-Leu₄)₂-Lys(Z)-OBzl. (SEQ.ID No. 5) The preferred peptide coupling agents are HOOBT and HBTU, thepreferred solvent is DMF and the preferred organic base is DIPEA. TheBoc protecting group is cleaved by treatingBoc-Leu-Leu-(Lys(Z)-Leu₄)₂-Lys(Z)-OBzl (SEQ. ID No. 5) with an acid atabout −20 to 0° C. under an inert atmosphere. The preferred acid was HCland the inert solvent was ethyl acetate.

[0014] H-Leu-Leu-(Lys(Z)-Leu₄)₂-Lys(Z)-OBzl (SEQ. ID No. 5) is treatedwith a peptide coupling agent, an organic base andBoc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) in an inert solvent atabout −4 to 10° C. to give Boc-Leu-Leu-(Lys(Z)-Leu₄)₃-Lys(Z)-OBzl. (SEQ.ID No. 3) The preferred peptide coupling agents are HOOBT and DIC, thepreferred solvent is THF and the preferred organic base is DIPEA. TheBoc protecting group is cleaved by treatingBoc-Leu-Leu-(Lys(Z)-Leu₄)₃-Lys(Z)-OBzl (SEQ. ID No. 3) with an acid atabout −20 to 0° C. under an inert atmosphere.

[0015] H-Leu-Leu-(Lys(Z)-Leu₄)₃-Lys(Z)-OBzl (SEQ. ID No. 3) was treatedwith a salt, an organic base, a peptide coupling agent, water andZ-Lys(Z)-Leu-Leu-OH in an inert solvent at about 0 to 20° C. over 2 to 5h to give Z-(Lys(Z)-Leu₄)₄-Lys(Z)-OBzl. (SEQ. ID No. 1) The preferredcoupling agents are DIC and HOOBT, the preferred solvent is THF and thepreferred salt is LiBF₄. The benzyl protecting groups are removed byhydrogenating Z-(Lys(Z)-Leu₄)₄-Lys(Z)-OBzl (SEQ. ID No. 1) in thepresence of a catalyst and an inert solvent under a positive H₂atmosphere. The preferred catalyst is Pd/C, the preferred solvent isacetic acid and the preferred H₂ pressure is 2 to 2.2 bar.

[0016] Procedure B follows another approach. This method employs aconvergent pathway where two larger fragments are combined to producethe protected final product. In addition this method usestetrabutylammonium hydroxide to saponify an ester protected carboxygroup with less than 1% racemization of the α-carbon.

[0017] As illustrated by Scheme B, H-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. IDNo. 2) is treated with Boc-Lys(Z)-Leu-Leu-OH, a peptide coupling agent,an organic amine in an inert solvent at about −5 to −2° C. for about 2to 3 h to give Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR. (SEQ. IDNo. 4) The preferred coupling agents are HOOBT and HBTU, the preferredorganic base is DIPEA, the preferred solvent is DMF and the preferredgroup for R is methyl. The terminal ester protecting group is saponifiedby treating Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 4)with a tetraalkylammonium hydroxide and water in an inert organicsolvent at about −17 to 0° C. over 10 to 160 min. Inert solvents includeDMF and THF, tetraalkylammonium hydroxide reagents includetetrabutylammonium hydroxide, tetramethylammonium hydroxide,tetraethylammonium hydroxide and tetrabenzylammonium hydroxide. Thepreferred solvent is THF, the preferred tetraalkylammonium hydroxide istetrabutylammonium hydroxide and the preferred temperature is −5 to 0°C. The reaction should be monitored by analytical methods, particularlyHPLC in order to determine when the starting ester is saponified, forracemization increases over time.

[0018] Boc-(Lys(Z)-Leu₄)Lys(Z)-Leu-Leu-OH (SEQ. ID No. 4) is treatedwith a salt, a peptide coupling agent, an organic base andH-Leu-Leu-(Lys(Z)Leu₄)₂-Lys-OBzl (SEQ. ID No. 5) in an inert organicsolvent at about 0 to 25° C. over 1 to 30 h to giveBoc-(Lys(Z)-Leu₄)₄Lys(Z)-OBzl. (SEQ. ID No. 1) The preferred couplingagents are HOOBT and DIC, the preferred organic base is DIPEA, thepreferred solvent is THF/water and the preferred salt istetrabutylammonium chloride.

[0019] The protecting groups of Boc-(Lys(Z)-Leu₄)₄Lys(Z)-OBzl (SEQ. IDNo. 1) are removed by treatment with trifluoroacetic acid followed byhydrogenation in the presence of a catalyst and an inert solvent under apositive H₂ atmosphere. The preferred catalyst is Pd/C, the preferredsolvent is acetic acid and the preferred H₂ pressure is 2 to 2.2 bar.

[0020] Although tetraalkylammonium hydroxides are employed in thesynthesis of (Lys-Leu₄)4-Lys, (SEQ. ID No. 1) their utility is notrestricted to the saponification of peptides with L-confirmations, norto peptides of Lys or Leu. It is used in the saponification ofAc-D-Nal-D-p-Cal-OMe, where this peptide was treated withtetraalkylammonium hydroxide in an inert solvent at about −10 to 0° C.to give Ac-D-Nal-D-p-Cal-OH. The preferred tetraalkylammonium hydroxideis tetrabutylammonium hydroxide and the preferred solvents are DMF andTHF. This procedure gives 2.6% of the undesired L isomer while othermethods, namely NaOH and aqueous acetone give 13% of the undesireddiastereomer. In addition, tetraalkylammonium hydroxides are used in thesaponification of Ac-D-Nal-D-p-Cal-D-3-Pal-L-Ser (OH)-OBzl. (SEQ. ID No.7) The preferred solvent for this saponification is THF, the preferredhydroxide is tetrabutylammonium hydroxide and the preferred temperatureis about −6 to −3° C.

[0021] The following examples are meant to illustrate the invention, notto limit it. Other embodiments will be obvious to those skilled in theart and are claimed by this invention. The identity of the compounds wasconfirmed by HPLC and LC comparison with known standards. The purity ofthe compounds was determined by their HPLC area %, where theracemization % was determined by the same method.

EXAMPLE 1 Step 1 Boc-Leu-Leu-OMe

[0022] 4-Methylmorpholine (12.62 g, 124.8 mmol), Boc-Leucine monohydrate(29.92, 120.0 mmol) and HOBt (1.62 g, 12.0 mmol) were added to asolution of H-Leu-OMe.HCl (21.80 g, 120.0 mmol) in ethyl acetate (205g). A solution of DCC (29.71 g, 144.0 mmol) in ethyl acetate (25.0 g)was added at 0 to 5° C. over 30 min and the resulting mixture wasstirred over 2 h at a maximum temperature of 20° C. The resulting ureawas removed by filtration and washed with ethyl acetate and the combinedorganic filtrate was extracted with successive portions of 5% aq. K₂CO₃and 5% aq. KHSO₄. The organic layer was dried (MgSO₄), concentrated invacuo, and the residual solid was recrystallized from petroleum ether(bp 100-125° C.) to giveBoc-Leu-Leu-OMe (38.6 g, 89.8%).

Step 2 Boc-Leu-Leu-OH

[0023] A solution of NaOH (1.34 g, 33.5 mmol) in water (15.0 g) wasadded dropwise over 10 min to the solution of Boc-Leu-Leu-OMe (10.0 g,27.9 mmol) in acetone (20.0 g) and water (50.0 g) at 18-22° C. Thereaction mixture was stirred at this temperature for 1 h and thenanalyzed by HPLC. When the methyl ester is under 5%, the reactionstirred for an additional 0.5h. A white turbidity formed by the end ofthe reaction which was removed by filtration and rinsed with water (5.0g). The clear filtrate was treated with formic acid (ca 0.8 g) to form acloudy solution followed by immediate crystallization of product.Additional formic acid (1.5 g) was added (to pH 4.0-4.5) and theresulting product was collected by filtration then was washed by amixture of acetone (10.0 g) and water (40.0 g). The solid product wassuspended in a mixture of acetone (14.0 g) and water (56.0 g) for 15min. The solid was collected by filtration and washed with a mixture ofacetone (7.5 g) and water (30.0 g) and dried in vacuo at 45° C. Yield:8.6-8.8 g (89.6-91.7%).

Step 3 H-Leu-Leu-OMe.HCl

[0024] Gaseous HCl (10.2 g, 279 mmol) was infused into a mixture ofBoc-Leu-Leu-OMe (17.9 g, 50.0 mmol) and ethyl acetate (71.4 g) at 20 to25° C. After 30 min, t-butyl methyl ether (142.9) was added to theresulting solution followed by a seed crystal. The resulting solidcrystalline product was filtered at 0 to 5° C. and dried in vacuo togive H-Leu-Leu-OMe.HCl (14.1 g, 95.7%).

Step 4 Boc-Lys(Z)-Leu-Leu-OMe

[0025] Isobutyl chloroformate (13.66 g, 100 mmol) was added dropwiseover 15 min at −15 to −10° C. to a solution of Boc-Lys(Z)-OH (38.04 g,100 mmol) in ethyl acetate (200 g). Another portion of ethyl acetate wasadded (10 g) and the mixture was stirred for 15 min. 4-Methylmorpholine(10.12 g, 100 mmol) was added dropwise over 15 min at −15 to −10° C.,followed by an additional portion of ethyl acetate (10.0 g) and theresulting mixture was stirred for 2 h. In a separate reaction vessel4-methylmorpholine (10.12 g, 100 mmol) was added dropwise over 15 min,to a solution of H-Leu-Leu-OMe.HCl (29.48 g, 100 mmol) in ethyl acetate(200.0 g) at −23 to −10° C. An additional portion of ethyl acetate (10.0g) was added followed by the addition of the formed anhydride over 30 to45 min at a temperature of −8 to −10° C. Another portion of ethylacetate (30.0 g) was added and the resulting mixture was stirred for 1.5h. Ethyl acetate (50.00 g) was added, the solution was filtered and theresulting organic filtrate was concentrated in vacuo. The resultingsolid was recrystallized from petroleum ether (bp 100 to 125) to giveBoc-Lys(Z)-Leu-Leu-OMe (55.9 g, 90.2%).

Step 5 H-Lys(Z)-Leu-Leu-OMe.HCl

[0026] A solution of HCl (28 g, 770 mmol) in ethyl acetate (100 g) at−10° C. was rapidly added to Boc-Lys(Z)-Leu-Leu-OMe (35.0 g, 55.74 mmol)under N₂ at −15 to −12° C. The mixture was stirred and maintained at −15to −12° C. for 60 to 70 min and allowed to crystallize for 1 h at −10 to−5° C. The solid was filtered while excluding moisture, washed withseveral portions of t-butyl methyl ether and dried in vacuo to giveH-Lys(Z)-Leu-Leu-OMe.HCl (28.7 g, 91.4%).

Step 6 Boc-Leu-Leu-Lys(Z)-Leu-Leu-OMe (SEQ. ID No. 2)

[0027] 4-Methylmorpholine (2.53 g, 25 mmol) was added to a solution ofH-Lys(Z)-Leu-Leu-OMe.HCl (13.93 g, 25 mmol) in DMF (85.0 g) at 10° C.HOBt (3.38 g, 25 mmol), Boc-Leu-Leu-OH (8.61 g, 25 mmol) and DIC (3.47g, 27.5 mmol) were added over 30 min at 8-10° C. An additional portionof DMF (5.0 g) was added and the reaction was kept at 8-10° C. for 16 h.The reaction was stirred at 18-20° C. for 3 h, cooled to 5-10° C. andtreated with H₂O (130.0 g). A solid precipitated at 25° C. which wasfiltered, washed with H₂O and recrystallized from MeOH and H₂O to giveBoc-Leu-Leu-Lys(Z)-Leu-Leu-OMe (SEQ. ID No. 2) (18.3 g, 86.4%).

Step 7 Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2)

[0028] A solution of NaOH (1.44 g, 36.0 mmol) in H₂O (15.0 g) was addedat 20-25° C. over 5 min to a mixture of Boc-Leu-Leu-Lys(Z)-Leu-Leu-OMe(SEQ. ID No. 2) (12.0 g, 14.16 mmol) THF (48.0 g) and H₂O (18.0 g). Theresulting mixture was stirred for 2 h. 98% Formic acid (5 g) was added,followed by ethyl acetate (30.0 g). The resulting organic phase waswashed with H₂O, concentrated in vacuo and the residue was dissolved inMeOH (60.0 g). The solids were filtered and rinsed with an additionalportion of MeOH (8.0 g). The combined MeOH solution was heated to 60-65°C. and H₂O (30.0 g) was added over 10 min. This mixture was cooled to20-25° C., the solid product began to crystallize. An additional portionof H₂O (35.0 g) was added and the mixture was stirred for 1 h. The solidproduct was filtered, washed with H₂O and dried in vacuo to giveBoc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) (11.5 g, 97.5%).

Step 8 Boc-Leu-Leu-Lys(Z)-OBzl

[0029] A solution of H-Lys(Z)-OBzl.HCl (20.35 g, 50 mmol) in DMF (50.0g) was treated with Et₃N (5.26 g, 51 mmol) at 25 to 30° C.Boc-Leu-Leu-OH (17.57 g, 51 mmol) and HOBt (1.08 g, 8 mmol) were addedfollowed by an additional portion of DMF (7.0 g). A solution of DCC(11.35 g, 55 mmol) in DMF (25.0 g) was added dropwise over a period of30 min at 20° C. The reaction mixture was stirred for 4 h and thetemperature was allowed to rise (to ca. 34° C.). Acetic acid (0.2 g) wasadded, the resulting suspension was filtered and the filter cake wasrinsed with DMF (15.0 g). Acetone (126.0 g) was added to the filtrate,followed by two portions of H₂O (66.5 g, 89.5 g-30 min apart). Theresulting precipitate was filtered and the filter cake was washed withacetone (53.0 g) and H₂O (212.0 g). The crude solid was triturated witha mixture of acetone (100 g) and H₂O (400g), filtered, washed with H₂Oand dried in vacuo at 40° C. to give Boc-Leu-Leu-Lys(Z)-OBzl (32.6 g,93.4%).

Step 9 H-Leu-Leu-Lys(Z)-OBzl

[0030] A cooled solution (−15° C.) of gaseous HCl (50.0 g, 1.37 mol) inethyl acetate (250 g) was added to Boc-Leu-Leu-Lys(Z)-OBzl (50.0 g, 71.7mmol) at −15 to −10° C. over 15 min. The mixture was stirred at −15 to−10° C. for 75 min and t-butyl methyl ether (310 g) was added over 25min. The temperature was allowed to rise to about −8 to −5° C. and theexcess HCl was removed under reduced pressure. Another portion oft-butyl methyl ether (310.0 g) was added over 15 min and the reactiontemperature was maintained at −5° C. for approximately 2 h. Theresulting solid product, H-Leu-Leu-Lys(Z)-OBzl.HCl was filtered andrinsed with t-butyl methyl ether then dried to give 41.8 g, 92.1%.

Step 10 Z-Lys(Z)-Leu-Leu-OMe

[0031] 4-Methylmorpholine (1.26 g, 12.5 mmol) and HOBt (1.69 g, 12.5mmol) were added to a solution of H-Leu-Leu-OMe.HCl (4.04 g, 13.75 mmol)in ethyl acetate (160.0 g) and DMF (36.0 g) over 15 min. Z-Lys(Z)-OH(5.18 g, 12.5 mmol) was added to this stirred mixture over 15 min. Asolution of DCC (3.10 g, 12.5 mmol) in ethyl acetate (10.0 g) was addeddropwise at 18-20° C. over 30 min. The reaction mixture was stirred at18-20° C. for 16 h and filtered. The filter cake was washed with ethylacetate (100.0 g) and the combined filtrate was washed with aqueousNaHCO₃. The resulting organic layer was washed with aqueous NaCl andconcentrated under reduced pressure. The residue was dissolved in MeOH(80.0 g) at about 50° C. Water (160 g) was slowly added and the mixturewas stirred for about 1 h at 20° C. The resulting solid was filtered,washed with H₂O (80.0 g) and dried at 40° C. in a vacuum oven to give8.16 g, 99.6% of the product Z-Lys(Z)-Leu-Leu-OMe.

Step 11 Z-Lys(Z)-Leu-Leu-OH

[0032] A solution of LiOH.H₂O (7.36 g, 0.182 mol) in H₂O (360 g) wasadded dropwise over 20 min to a solution of Z-Lys(Z)-Leu-Leu-OMe (100 g,0.152 mol) in acetone (40 mL) under N₂ at 25° C. The reaction mixturewas stirred for an additional 40 min at 25° C. The pH was adjusted to 4,using 96% formic acid (1.25 g, 0.0273 mol). The reaction mixture wascooled to 15° C. and water (42 g) was added dropwise over 1 h followedby a few seed crystals of Z-Lys(Z)-Leu-Leu-OH and the mixture wasstirred at 15° C. for 2 h. The resulting solid was collected byfiltration and washed with H₂O (10 mL) and acetone (10 mL). The solidwas air dried then further dried in a vacuum oven at 45° C. for 24 h togive 87.3 g, 89.7% of Z-Lys(Z)-Leu-Leu-OH.

Step 12 Boc-LLK(Z)-LLLLK(Z)-OBzl (SEQ. ID No. 6)

[0033] Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) (41.65 g, 50 mmol),H-Leu-Leu-Lys(Z)-OBzl.HCl (31.65, 50.0 mmol) HOBt (8.10 g, 60 mmol), andHBTU (22.75, 60 mmol) was dissolved in DMF (200 g) and acetonitrile (300g) and was cooled to 0° C. DIEA (19.35 g, 150 mmol) was added over 10min and the resulting mixture was stirred at 0° C. for 2 h. Thetemperature was allowed to rise to 20° C. and the mixture was stirredfor another 1 h. The reaction mixture was poured into water (2500 g) andthe resulting solid was collected by filtration and dried in vacuo at45° C. The crude solid was powdered and stirred with a mixture ofacetone (500 g) and t-butyl methyl ether (500 g) for 20 min at 50° C.and for 1.5 h at room temperature. This mixture was filtered and thesolid was washed with a mixture of acetone (50 g) and t-butyl methylether (50 g) to give Boc-LLK(Z)-LLLLK(Z)-OBzl (SEQ. ID No. 6) (65,6 g,92.9%).

Step 13 H-LLK(Z)-LLLLK(Z)-OBzl.TFA (SEQ. ID No. 6)

[0034] Trifluoroacetic acid (11 mL) was cooled in an ice bath to 0° C.under N₂. Boc-LLK(Z)-LLLLK(Z)-OBzl (SEQ. ID No. 6) (2.5 g, 1.77 mmol)was added in one portion and the mixture stirred at 0° C. for 1 h. Theresulting solution was poured into ice water (100 mL) and stirred for 20min. The resulting white solid was collected by filtration and washedwith distilled water (50 mL) then air-dried. Yield of white solidproduct was 2.49 g (98.8%); HPLC area % purity: 97%.

Step 14 Boc-LL-K(Z)LLLLK(Z)-LLLLK(Z)-OBzl (SEQ. ID No. 5)

[0035] H-LLK(Z)LLLLK(Z)-OBzl.TFA (SEQ. ID No. 6) (19.4 g, 13.608 mmol)was dissolved in DMF (500 mL) at 50° C. in a 1L reaction flask. Thesolution was cooled to −4° C. and stirred under nitrogen.Boc-LLK(Z)LL-OH (SEQ. ID No. 2) (11.60 g, 13.92 mmol) was added and wasdissolved immediately. HOBT (4.44 g, 27.216 mmol) and HBTU (6.193 g,16.3 mmol were added followed by a solution of DIPEA (5.28 g, 40.824mmol) in DMF (20 mL) at −4° C. over 10 min. The resulting yellowsolution was stirred at −4° C. for 4 h (HPLC analysis showed a nearcomplete reaction after 2 h). The reaction mixture was allowed to warmup to 10° C. then was poured into ice water (1L). Saturated aq. NaCl(100 mL) was added and the mixture stirred for 20 min at rt. The yellowsolid was collected by filtration and was washed with distilled water(300 mL) then was air-dried overnight. Yield of crude product (28.9 g).The crude solid was suspended in MeOH (200 mL) where it became sticky.The mixture was heated to boiling on a steam bath; the materialsolidified as a result of this treatment. The lumpy solid was powderedand heating was continued for additional 10 min. The mixture was allowedto cool to room temperature and the solid was collected by filtration,rinsed with MeOH (50 mL) and dried to giveBoc-LL-K(Z)LL*LLK(Z)-LLLLK(Z)-OBzl (SEQ. ID No. 5) (27.9 g, 96.5%), area% HPLC purity: 93.0%, D-13 (*) diastereomer: 0.4%.

Step 15 H-LLK(Z)LLLLK(Z)-LLLLK(Z)-OBzl.HCl (SEQ. ID No. 5)

[0036] Gaseous HCl (98.6 g) was introduced to a suspension ofBoc-LLK(Z)LLLLK(Z)-LLLLK(Z)-OBzl (SEQ. ID No. 5) (38.1 g, 17.9 mmol) inethyl acetate (280 g) at −18 to −22° C. over 80 min. The resultingsolution was stirred at −16 to −14° C. for 60 min. The excess HCl wasremoved under reduced pressure at −20 to −0° C. and diethyl ether (192g) was added at −3 to 3° C. A solution of potassium bicarbonate (33 g)in H₂O (200 g) was added in portions until the pH of the reactionmixture was between 1.5 and 2.5, the resulting mixture was warmed to 19to 23° C. and stirred for 30 min. The resulting solid was collected byfiltration, rinsed with H₂O and dried in vacuo at 35 to 40° C. to giveH-LLK(Z)LLLLK(Z)-LLLLK(Z)-Obzl.HCl (SEQ. ID No. 5) (34.9 g, 95%).

Step 16 Boc-LLK(Z)LLLLK(Z)LLLLK(Z)-LLLLK(Z)-OBzl (SEQ. ID No. 3)

[0037] H-LLK(Z)LLLLK(Z)-LLLLK(Z)-OBzl (SEQ. ID No. 5) (10.0 g, 4.85mmol) was added to a stirred solution of LiBF₄ (3.04 g, 32.4 mmol) inNMP (99 g) THF (286 g). Boc-Leu-Leu-K(Z)Leu-Leu-OH (5.24 g, 6.29 mmol)was added and the mixture was homogenized until a viscous gel wasformed. Water (36 g) was added and the mixture was stirred for 20 min awell stirred turbid solution is obtained. The reaction mixture wascooled to −6 to −4° C. and treated with HOOBt (2.76 g, 16.92 mmol) andDIC (1.84 g, 14.58 mmol). DIPEA (3.76 g, 29.09 mmol) in THF (4 g) wasadded to the resulting solution while maintaining an internaltemperature of −6 to −4° C. The resulting mixture was stirred at −6 to−4° C. for 1 h and allowed to warm to 20 to 25° C. over 3 h then stirredat this temperature overnight. THF was removed in vacuo at 35-40° C. andthe remaining mixture was diluted with MeOH (20 g). This resultingyellow solution was added dropwise, over 5-10 min, to a cooled solutionof Na₂CO₃ (3.34 g) in H₂O (330 g) and rinsed with MeOH (5 g). Theresulting mixture was stirred for 15 min and the precipitate wascollected by filtration. This crude solid was suspended in acetonitrile(110 g), warmed to 60-70° C., and the fine suspension was added to water(48 g) and cooled to 20-25° C. The resulting solid was separated, rinsedwith a solution of H₂O (24 g) and acetonitrile (28 g) and dried undervacuum at 38-42° C. to give Boc-LLK(Z)LL*LLK(Z)LLLLK(Z)-LLLLK(Z)-OBzl(SEQ. ID No. 3) (12.05 g, 89%), D-8 (*) diastereomer: 5.5%.

Step 17 H-LLK(Z)LLLLK(Z)LLLLK(Z)-LLLLK(Z)-OBzl.TFA (SEQ. ID No. 3)

[0038] Trifluoroacetic acid (180 g) was cooled to −10 to −14° C. Thepowdered solid Boc-LLK(Z)LLLLK(Z)LLLLK(Z)LLLLK(Z)-OBzl (SEQ. ID No. 3)(50 g, 17.6 mmol) was added slowly into the cooled TFA. The mixturestirred at −9 to −5° C. for 3 h or until complete dissolution. Ethanol(99 g) was added slowly at −9 to −5° C. The resulting cold solution wasadded slowly to a solution of KHCO₃ (197.5 g, 507 mmol) in water (750 g)cooled to 12-18° C. The white solid was collected by filtration andwashed with water (480 g). The wet solid was slurried in water (500 g)for 10 min then filtered and washed with water (50 g). The white solidwas dried under reduced pressure at 40-45° C. to giveH-LLK(Z)LLLLK(Z)LLLLK(Z)-LLLLK(Z)-OBzl.TFA (SEQ. ID No. 3) (51.16 g,102%), HPLC purity: 88.2%.

Step 18 Z-K(Z)LLLLK(Z)LLLLK(Z)LLLLK(Z)-LLLLK(Z)-OBzl (SEQ. ID No. 1)

[0039] Finely powdered H-LLK(Z)LLLLK(Z)LLLLK(Z)LLLLK(Z)-OBzl.TFA (SEQ.ID No. 3) (23.13 g, 8.1 mmol) was added slowly to a mixture of NMP (40g) and THF (160 g). This was followed by the addition of Z-K(Z)LL-OH(5.71 g, 8.9 mmol). The resulting solution was cooled to −3 to 3° C.HOOBt (3.96 g, 24.3 mmol) and HBTU (3.99 g, 10.5 mmol) were addedfollowed by NMP (for rinse) and the mixture stirred for a few minutesuntil complete dissolution then cooled to −6 to −9° C. A solution ofDIPEA (4.19 g, 32.4 mmol) in THF (4 g) was added slowly at −6 to −9° C.leading to a deep orange color. The solution was stirred for 2 h at −6to −9° C., for 3h at −1 to 3° C. and then was warmed up slowly to roomtemperature. The THF was distilled under reduced pressure at 40-45° C.to give an oily yellow residue. The residue was added to a solution ofNa₂CO₃ (1.8 g) at 15-25° C. The resulting suspension was stirred for 15min and the solid was isolated by filtration and washed with water (75g) then with a mixture of water (26 g) and iPrOH (32 g). The wet crudesolid was slurried in a mixture of iPrOH (104 g) and water (86 g) for 30min at 20-25° C. The solid was collected by filtration, washed with amixture of iPrOH (27 g) and water (22 g) and dried in vacuo at 38-42° C.to give Z-K(Z)LL*LLK(Z)LLLLK(Z)-LLLLK(Z)-LLLLK(Z)-OBzl (SEQ. ID No. 1)(25.1 g, 92%), HPLC purity: 90%, D-3 (*) diastereomer: 1.1%.

Step 19 (Lys-Leu₄)₄-Lys (SEQ. ID No. 1)

[0040] Z-K(Z)LLLLK(Z)LLLLK(Z)LLLLK(Z)-LLLLK(Z)-OBzl (SEQ. ID No. 1) (630g, 187.26 mmol) was added to a mixture of trifluoroacetic acid (2815 g)and acetic acid (3969 g). After dissolution, water (630 g) was addedfollowed by wet (50% H₂O) 5% Pd/C (252 g). The mixture was hydrogenatedunder a H₂ pressure of about 2.8 bar for 3 h. The mixture was filteredin absence of air and was rinsed with a mixture of TFA (939 g), AcOH(1323 g) and H₂O (210 g). The filtrate was concentrated at <35° C. underreduced pressure to give an oil. This oil was dissolved in iPrOH (2021g) and added dropwise, within 30 min into stirred t-butyl methyl ether(12405 g) and rinsed with iPrOH (300 g). After stirring for 30 min atrt, the solid was collected by filtration and was washed, immediatelywith t-butyl methyl ether (18173 g) and dried in a vacuum oven at <40°C. to give crude (Lys-Leu₄)₄-Lys. (SEQ. ID No. 1) Yield: 534 g, 94.7%,HPLC area % purity: 81.8%.

EXAMPLE 2 Step 1 Boc-Lys(Z)-Leu-Leu-OH

[0041] Boc-Lys(Z)-Leu-Leu-OMe (12.0 g, 19.33 mmol) was dissolved inacetone (41.0 g, 52 mL) at 22-23° C. A solution of NaOH (1.94 g, 48.5mmol) in water (18.0 g) was added over 5 min. After stirring for 40min., the reaction was quenched with a solution of citric acid (10.2 g,53.0 mmol) in water (18.0 g). The acetone was removed on a rotaryevaporator at 40° C. The remaining mixture was extracted with ethylacetate (2×65 mL). The EtOAc extract was washed with water (50 g) thenwith aqueous saturated sodium chloride solution (50 g). The EtOAcextract was dried over anhydrous MgSO₄ (10 g). The EtOAc solution wasfiltered and diluted with DMF (40. g). The ethyl acetate was evaporatedat 40° C. under reduced pressure to give the solution of the product inDMF. Weight of solution ˜52 g.

Step 2 H-Leu-Leu-Lys(Z)-Leu-Leu-OMe.HCl (SEQ. ID No. 2)

[0042] Boc-Leu-Leu-Lys(Z)-Leu-Leu-OMe (SEQ. ID No. 2) (18.2 g, 21.5mmol) was added to a solution of HCl gas (31.0 g, 850 mmol) in MeOH (360g) and cooled to 5° C. The reaction mixture was stirred, allowed to warmup to room temperature and stirring was continued for 4.5 h. Thereaction mixture was cooled to 0° C., poured into ice/H₂O (630 g) andstirred for 1 h. The white solid was collected by filtration, washedwith distilled water (500 mL) and air dried. Yield of product: 14.8 g,87.6%; HPLC area % purity: 99.2%.

Step 3 Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OMe (SEQ. ID No. 4)

[0043] H-Leu-Leu-Lys(Z)-Leu-Leu-OMe.HCl (SEQ. ID No. 2) (15.12 g, 19.33mmol) was dissolved in DMF (100 g). A solution of 11.73 gBoc-Lys(Z)-Leu-Leu-OH (19.33 mmol) in DMF (obtained from step 20) wasadded and the resulting solution was cooled to −5° C. while stirringunder nitrogen. To the cold solution was added HOOBt (3.30 g, 20.2 mmol)followed by HBTU (7.67 g, 20.2 mmol) and DIPEA (7.76 g, 60.03 mmol) over5 min. The clear yellow solution became cloudy after about 5 min. Thereaction mixture stirred at −4° C. for 80 min. The mixture becamethicker as the reaction progressed but was easy to stir. The mixture wasquenched by adding a solution of K₂CO₃ (10.0 g) in water (200 g). Themixture warmed up gently on a steam bath to 40-45° C. and was kept atthat temperature for about 30 min. The solid was collected by filtrationand washed with hot (60-70° C.) water (2×150 g). The product was driedovernight in a vacuum oven at 40° C. to giveBoc-Lys(Z)-Leu₄Lys(Z)-Leu-Leu-OMe (SEQ. ID No. 4) (25.0 g, 96.8%). HPLCarea % purity: 94%.

Step 4 Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 4)

[0044] Boc-Lys(Z)-Leu₄Lys(Z)-Leu-Leu-OMe (SEQ. ID No. 4) (16.03 g,12.0mmol) was powdered and suspended in THF (240.0 g, 270 mL). The mixturestirred and cooled to −4° C. under nitrogen. The cold solution wastreated with 40% aqueous tetrabutylammonium hydroxide (24.0 g, 37.0mmol), added dropwise over 5 min. (the temperature maintained at <−1° C.during the addition). The suspension dissolved to form a clear lightyellow solution. The HPLC analysis indicated a complete hydrolysis after50 min. The reaction mixture stirred at −2° C. for 70 min. and wasquenched with a solution of glacial acetic acid (6.0 g, 100.0 mmol) inwater (9.0 g). The reaction mixture remained as a clear solution. Thereaction flask was equipped with an addition funnel containing anaqueous solution made of saturated aqueous sodium chloride (50.0 g) andwater (200 g). The initial volume of the solution in the reaction flaskwas marked. The reaction mixture was heated to distill the THF atatmospheric pressure while adding aqueous NaCl to maintain a constantvolume. Precipitation started about midway of the evaporation/addition.Near the end of evaporation foaming occurred. At that point, heatingstopped and the remaining aqueous solution was added. The temperaturereached 83° C. at the end of evaporation and a total of 224 g of THFdistillate was collected. The hot mixture was centrifuged to collect thewhite solid product. The solid was washed with a total of hot (50° C.)water (200 g). The white solid was dried at 40° C. in a vacuum oven togive Boc-Lys(Z)-Leu-Leu*-Leu-Leu-Lys(Z)-Leu-Leu**-OH (SEQ. ID No. 4)(15.6 g, 98%), HPLC area % purity: 91.4%. diasteromeric purity: 99.08%,D3-diastereomer(*): 0.42%, D8-diastereomer(**): 0.50%.

Step 5 Boc-(Lys(Z-Leu₄)₄Lys(Z)-OBzl (SEQ. ID No. 1)

[0045] Boc-K(Z)LLLLK(Z)LL-OH (SEQ. ID No. 4) (6.61 g, 5.0 mmol) wasmixed with H-LLK(Z)LLLLK(Z)-LLLLK(Z)-OBzl.HCl (SEQ. ID No. 5) (10.32 g,5.0 mmol) and 85% Bu₄NCl (7.5 g, 22.9 mmol) in tetrahydrofuran (400 g)and purified water (30 g). The mixture was cooled to 0° C. and treatedwith HOOBt (2.25 g, 15.0 mmol) and N,N-diisopropylcarbodiimide (DIC)(1.90 g, 15.0 mmol), followed by N,N-diisopropyl ethylamine (DIPEA)(2.25 g, 17.4 mmol). The yellow solution was stirred at 0° C. for 1 hthen at rt (23-25° C.) for 20 h and finally at 30° C. for 2 h. Thereaction mixture became a clear yellow solution in a few hours from thestart of reaction. At the end of the 24 h period, the yellow solutionwas diluted with 1-methyl-2-pyrrolidone (100 g) and the THF wasevaporated at 35-40° C. under reduced pressure. The residual solutionwas added to cold water (350 g) containing potassium carbonate (12 g).The flask was rinsed with methanol (50 mL) and the rinse was combinedwith the aqueous mixture. The product precipitated as a fine yellowsolid. The mixture stirred slowly at rt for 20-30 min and the solid wascollected by filtration and was washed with water (150 mL) followed byan 80:20 MeOH/H₂O mixture (250 mL). The solid was air-dried on thefiltration funnel for about an hour then further dried in a vacuum ovenat 40° C. overnight. The product was a light yellow solid, 16.6 g(99.7%), HPLC area % purity: 92%.

Step 6 (Lys-Led₄)₄Lys (SEQ. ID No. 1)

[0046] Boc-(Lys(Z)-Leu₄)₄Lys(Z)-OBzl (SEQ. ID No. 1) (16.0 g, 4.8 mmol)was dissolved in trifluoroacetic acid (64.0 g) at room temperature. Ittook about 20 -25 min for the solid to completely dissolve. Glacialacetic acid (80.0 g) was added followed by distilled water (16.0 g) andfinally 5% palladium on moist activated carbon (4.5 g). The mixture washydrogenated at 40-50 psi overnight (about 18 h). The resulting mixturewas filtered through a Buchner funnel (5.5 cm diameter) using fiveWhatmann qualitative filter papers. The hydrogenation flask was rinsedwith trifluoroacetic acid (10.0 g). The colorless filtrate wasconcentrated under reduced pressure at 40-45° C. to a weight of about 30g. The residue was dissolved in trifluoroacetic acid (74.0 g) and wasadded slowly over 10 min to t-butyl methyl ether (300 mL) while coolingin an ice bath with stirring. The product precipitated as a white solid.After stirring for about 15 min., the product was collected byfiltration and was washed immediately with t-butyl methyl ether (100mL). The solid was dried in a vacuum oven at 40° C. overnight. Yield ofisolated solid: 14.74 g (95.6% assuming the formation of ahexatrifluoroacetate salt), HPLC area % purity: 80.5%.

EXAMPLE 3 Ac-D-Nal-D-p-Cal-OH

[0047] Ac-D-Nal-D-p-Cal-OMe (45.3 g, 100.0 mmol), powdered to a finesolid, was suspended and stirred in THF (450.0 mL) then cooled to −6° C.The cold suspension was treated with 40% aqueous tetrabutyl ammoniumhydroxide (72.0 g, 111.0 mmol), added in such a rate that the reactiontemperature did not exceed −3° C. The solid dissolved as the base wasadded and was a completely in solution at the end of addition. Thereaction was complete shortly after the end of addition (as determinedby HPLC analysis). The reaction was acidified at −6° C. by a slowaddition of a solution of conc. HCl (20 mL) in water (20 mL). Afteracidification, the clear solution was diluted with water (100 mL) andmost of the THF was evaporated on a rotovap at 25-30° C. until heavyprecipitation occurred. The mixture was diluted with methanol (200 mL)and the solid was collected by filtration. The flask and the solid wererinsed with additional methanol (150 mL) and the solid was air-dried.Yield of isolated dry solid: 40.2 g, 91.6%. HPLC area % purity: 93.7%,diastereomer (*): 2.6%, Ac-D-Nal-D-p-Cal*-OH

EXAMPLE 4 AC-D-Nal-D-pCal-D-3-Pal-L-Ser (OH)-OH (SEQ. ID No. 7)

[0048] Ac-D-Nal-D-p-Cal-D-3-Pal-L-Ser (OH)-OBzl (SEQ. ID No. 7) (58.1 g,76.0 mmol), powdered to a fine solid, was suspended in THF (400.0 mL)then stirred and cooled to −5° C. The cold suspension was treated with40% aqueous tetrabutylammonium hydroxide (98.6 g, 152.0 mmol), added insuch a rate that the reaction temperature did not exceed −2° C. (about15 min). The solid dissolved slowly after the base was added andcompletely dissolved after 40 min following the end of addition. Thereaction mixture stirred for a total of 1.5 h, during which thetemperature was allowed reach and remain at 0° C. The HPLC analysisindicated a complete hydrolysis. The resulting clear solution was pouredinto a solution of glacial acetic acid (35 g) in ice/water (1.1 L) withstirring. The product precipitated as a thick white solid which wascollected by filtration. The wet cake was slurried in hot (65° C.) water(1 L) and filtered. The wet cake was slurried in methanol (700 mL) andheated to boiling with stirring. The solid was collected by filtrationfrom the hot slurry and was rinsed with methanol (150 mL) then wasair-dried. Yield of isolated dry powdered solid: 44.8 g, 87.4%. HPLCarea % analysis of product: 95%, diastereomer (*): 1.7%,Ac-D-Nal-D-p-Cal-D-3-Pal-L-Ser*(OH)-OH. (SEQ. ID No. 7)

What is claimed is:
 1. A process for producing the synthetic KL-4pulmonary surfactant protein of the formula (Lys-Leu₄)₄Lys (SEQ. IDNo. 1) comprising the steps of: a. reacting a 3-amino acid peptideresidue of the formula H-Lys(Z)-Leu-Leu OH with a 5-amino acid peptideresidue Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) to yield an8-amino acid peptide residue, which is successively reacted with the5-amino acid peptide to form an 18-amino acid peptide of the formulaH-Leu-Leu (Lys(Z)-Leu₄)₃Lys(Z)-OBzl (SEQ. ID No. 3); b. reacting the 18amino acid peptide residue with a 3-residue amino acid peptide of theformula: H-Leu-Leu-Lys(Z)-OBzl to form the protected 21-amino acid KL-4protein; and c. removing the protecting group of the 21-amino acid KL-4protein by reaction a suitable acid to form the final KL-4-protein.
 2. Aprocess for producing (Lys-Leu₄)₄Lys (SEQ. ID No. 1) comprising thesteps of: a. reacting H-Leu-Leu-Lys(Z)-OBzl,Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2), and a peptide couplingagent in an inert organic solvent, to form:Boc-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6); b.reacting Boc-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6)with a suitable acid with or without an inert organic solvent to form:H-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6); c.reacting H-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6),Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) and a peptide couplingagent in an inert organic solvent to form:Boc-Leu-Leu-(Lys(Z)-Leu₄)₂-Lys(Z)-OBzl (SEQ. ID No. 5); d. reactingBoc-Leu-Leu-(Lys(Z)-Leu₄)₂-Lys(Z)-OBzl (SEQ. ID No. 5) with a suitableacid, with or without an inert organic solvent to form:H-Leu-Leu-(Lys(Z)-Leu₄)₂-Lys(Z)-OBzl (SEQ. ID No. 5); e. reacting,Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2), a peptide coupling agent,a saltand H-Leu-Leu-(Lys(Z)-Leu₄)₂-Lys(Z)-OBzl (SEQ. ID No. 5) in aninert solvent to form: Boc-Leu-Leu-(Lys(Z)-Leu₄)₃-Lys(Z)-OBzl (SEQ. IDNo. 3); f. reacting Boc-Leu-Leu-(Lys(Z)-Leu₄)₃-Lys(Z)-OBzl (SEQ. ID No.3) with a suitable acid, with or without an inert organic solvent toform: H-Leu-Leu-(Lys(Z)-Leu₄)₃-Lys(Z)-OBzl (SEQ. ID No. 3); g. reactingZ-Lys(Z)-Leu-Leu-OH, water, a peptide coupling agent andH-Leu-Leu-(Lys(Z)-Leu₄)₃-Lys(Z)-OBzl (SEQ. ID No. 3) in an inert organicsolvent to form: Z-(Lys(Z)-Leu₄)₄-Lys(Z)-OBzl (SEQ. ID No. 1); h.reacting, acetic acid, water, a catalyst andZ-(Lys(Z)-Leu₄)₄-Lys(Z)-OBzl (SEQ. ID No. 1) under an H₂ atmosphere of 2to 2.2 bar to form: (Lys-Leu₄)₄Lys (SEQ. ID No. 1).
 3. The processaccording to claim 2(a), wherein the peptide coupling agent isHOOBT/HBTU and the inert organic solvent is DMF
 4. The process accordingto claim 2(b), wherein the suitable acid is TFA and an organic solventis absent.
 5. The process according to claim 2(c), wherein the peptidecoupling agent is HOOBT/HBTU and the inert organic solvent is DMF. 6.The process according to claim 2(d), wherein the inert organic solventis ethyl acetate and the suitable acid is gaseous HCl.
 7. The processaccording to claim 2(e), wherein the peptide coupling agent isHOOBT/HBTU, the salt is LiBF₄ and the inert organic solvent is DMF. 8.The process according to claim 2(f), wherein the inert solvent is EtOHand the suitable acid is trifluoroacetic acid.
 9. The process accordingto claim 2(g), where the peptide coupling agent is HOOBT/HBTU, and theinert organic solvent is DMF.
 10. The process according to claim 2(h),wherein the catalyst is 5% Pd/C.
 11. A process for producing(Lys-Leu₄)₄Lys (SEQ. ID No. 1) comprising the steps of: a. reacting acompound of the formula Boc-Lys(Z)Leu-Leu-OH and a peptide couplingagent with a compound of the formula: H-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ.ID No. 2), a suitable organic solvent to form a compound of the formula:Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 4), wherein Ris C₁₋₈alkyl or phenylC₁₋₈alkyl; b. removing the carboxy protectinggroup by reacting a hydroxide, salt, water andBoc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 4) in an inertorganic solvent, to form: Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OH(SEQ. ID No. 4); c. reactingBoc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 4),H-Leu-Leu-(Lys(Z)-Leu₄)₂-Lys(Z)-OBzl (SEQ. ID No. 5), a salt, water anda peptide coupling agent in an inert organic solvent, to form:Boc-(Lys(Z)-Leu₄)₄-Lys(Z)-OBzl (SEQ. ID No. 1); d. reactingtrifluoroacetic acid, Boc-(Lys(Z)-Leu₄)₄-Lys(Z)-OBzl (SEQ. ID No. 1),acetic acid, water and a catalyst under a H₂ atmosphere of 40 to 50 psito form (Lys-Leu₄)₄Lys (SEQ. ID No. 1).
 12. The process according toclaim 11 (a) wherein, the peptide coupling agent is HOOBT/HBTU, and theinert organic solvent is DMF
 13. The process according to claim 11(b)wherein, the hydroxide is tetrabutylammonium hydroxide and the inertsolvent is THF or DMF.
 14. The process according to claim 11 (c)wherein, the peptide coupling agent is HOOBT/DIC, the salt istetrabutylammonium chloride and the inert organic solvent is DMF. 15.The process according to claim 11(d) wherein, the catalyst is Pd/C. 16.Intermediates useful in processes for the production of (Lys-Leu₄)₄Lys(SEQ. ID No. 1) selected from the group consisting of:H-Leu-Leu-Lys(Z)-OBzl, Boc-Lys(Z)-Leu-Leu-OR, Z-Lys(Z)-Leu-Leu-ORwherein R is C₁₋₈alkyl or phenylC₁₋₈alkyl and suitable acid saltsthereof.
 17. Intermediates useful in processes for the production of(Lys-Leu₄)₄Lys (SEQ. ID No. 1) selected from the group consisting of:Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH, (SEQ. ID No. 2)Boc-Leu-Leu-Lys(Z)-Leu₄-Lys(Z)-OBzl (SEQ. ID No. 6),H-Leu-Leu-Lys(Z)-LeU₄-Lys(Z)-OBzl (SEQ. ID No. 6),Boc-Leu-Leu-(Lys(Z)-Leu₄)₂-Lys(Z)-OBzl (SEQ. ID No. 5)H-Leu-Leu-(Lys(Z)-Leu₄)₂-Lys(Z)-OBzl (SEQ. ID No. 5),Boc-Leu-Leu-(Lys(Z)-Leu₄)₃-Lys(Z)-OBzl (SEQ. ID No. 3),H-Leu-Leu-(Lys(Z)-Leu₄)₃-Lys(Z)-OBzl (SEQ. ID No. 3),-(Lys(Z)-Leu₄)₄-Lys(Z)-OBzl, Boc-Leu-Leu-Lys(Z)-Leu₄-Lys(Z)-Leu-Leu-OH(SEQ. ID No. 4), wherein Z is benzyloxycarboxyl and suitable acid saltsthereof.
 18. A method of saponifying an ester protected carboxy group ofa peptide, which consists or reacting said protected peptide with atetraalkylammonium hydroxide and water in an inert organic solvent at alow temperature to form the deprotected carboxy group.
 19. The method ofclaim 18 wherein, the tetraalkylammonium hydroxide is tetrabutylammoniumhydroxide, the inert organic solvent is DMF or THF and the temperatureis about −20 to 0° C.
 20. A process for coupling polypeptide fragmentshaving 8-25 amino acid residues by reacting said fragments with asuitable peptide coupling agent in the presence of a tetraalkylammoniumhalide as a solubilizing agent.
 21. A method of saponifyingAc-D-Nal-D-p-Cal-OMe which consists of treating this peptide with atetraalkylammonium hydroxide and water in an inert organic solvent at alow temperature to form Ac-D-Nal-D-p-Cal-OH.
 22. The method of claim 21wherein, the tetraalkylammonium hydroxide is tetrabutylammoniumhydroxide, the inert organic solvent is DMF or THF and the temperatureis about −20 to 0° C.
 23. A method of saponifyingAc-D-Nal-D-p-Cal-D-3-Pal-L-Ser (OH)-OBzl (SEQ. ID No. 7) which consistsof treating this peptide with a tetraalkylammonium hydroxide and waterin an inert organic solvent at a low temperature to formAc-D-Nal-D-p-Cal-D-3-Pal-L-Ser (OH)-OH (SEQ. ID No. 7).
 24. The methodof claim 23 wherein, the tetraalkylammonium hydroxide istetrabutylammonium hydroxide, the inert organic solvent is DMF or THFand the temperature is about −20 to 0° C.